The modification of DNA in eukaryotes by methylation has regulatory effects on gene regulation.
Cancer treatments, in general, have a higher rate of success if the cancer is diagnosed early and treatment is started earlier in the disease process. The relationship between improved prognosis and stage of disease at diagnosis hold across all forms of cancer for the most part. Therefore, there is an important need to develop early assays of general tumorigenesis through marker assays that measure general tumorigenesis without regard to the tissue source or cell type that is the source of a primary tumor. Moreover, there is a need to address distinct genetic alteration patterns that can serve as a platform associated with general tumorigenesis for early detection and prognostic monitoring of many forms of cancer.
Importance of DNA Methylation
Methylation of DNA is a mechanism for changing the sequence of DNA without altering its coding function. DNA methylation is a heritable, reversible and epigenetic change. DNA methylation harbours the potential to alter gene expression which in turn affects developmental and genetic processes. The methylation reaction involves flipping a target cytosine out of an intact double helix thereby allowing the transfer of a methyl group from S-adenosylmethionine in a cleft of the enzyme DNA (cytosine-5)-methyltransferase (Klimasauskas et al., Cell 76:357-369, 1994) to form 5-methylcytosine (5-mCyt). This enzymatic conversion is the only epigenetic modification of DNA known to exist in vertebrates and is essential for normal embryonic development (Bird, Cell 70:5-8, 1992; Laird and Jaenisch, Human Mol. Genet. 3:1487-1495, 1994; and Bestor and Jaenisch, Cell 69:915-926, 1992).
CpG-rich sequences are known as CpG islands (1). CpG islands are distributed across the human genome and often span the promoter region as well as the first exon of protein coding genes. Methylation of individual promoter region CpG islands usually turns off or reduce the rate of transcription by recruiting histone deacetylases, which supports the formation of inactive chromatin (2). CpG islands are typically between 0.2 to about 1 kb in length and are located upstream of many housekeeping and tissue-specific genes, but may also extend into gene coding regions. Therefore, it is the methylation of cytosine residues within CpG islands in somatic tissues, which is believed to affect gene function by altering transcription (Cedar, Cell 53:3-4, 1988).
Abnormal methylation of CpG islands associated with tumor suppressor genes may also cause decreased gene expression. Increased methylation of such regions may lead to progressive reduction of normal gene expression giving abnormal cells a growth advantage (i.e., a malignancy).
Methylation promoter regions, particularly in tumour suppressor genes, and genes involved in apoptosis and DNA repair, is one of the hallmarks of cancer (2). Changes in the methylation status of these genes are an early event in cancer and continue throughout the different stages of the cancer. Specifically, distinct tumour types often have characteristic patterns of methylation, which can be used as markers for early detection and/or monitoring the progression of carcinogenesis (3, 4). For therapeutic purposes, the methylation of certain genes, particularly DNA repair genes, can cause sensitivity to specific chemotherapeutics and methylation of those genes can thereby act as a predictive marker if those chemotherapeutic agents are used (5).
A number of current methodologies for methylation studies already exist (9). Sequencing of bisulphite-treated DNA is the gold standard for methylation studies as it reveals directly the status of each CpG dinucleotide. The technique is, however, both time consuming and cost inefficient, and therefore not immediately applicable for large scale analysis such as screening programmes.
Another technique is the methylation specific PCR (MSP) (21). U.S. Pat. No. 5,786,146 discloses a method of methylation specific PCR (MSP) for identifying DNA methylation patterns in a CpG containing nucleic acid. The method uses agents to modify unmethylated cytosine in the nucleic acid. CpG specific oligonucleotide primers are used to distinguish between modified methylated and unmethylated nucleic acid. The identification of the methylated nucleic acid is based on the presence or absence of amplification product resulting from the amplification and distinguishing modified methylated and non-methylated nucleic acids. However, methylation-specific PCR (MSP) is prone to false positive results and may result in overestimation of the number of methylated samples. Furthermore, MSP mainly offers a qualitative result of the methylation status of the target nucleic acid. The results are not easily quantified.
An alternative methodology for determination of methylation status is methylation-sensitive melting curve analysis (MS-MCA) or high resolution melting curve analysis (HRMS-MCA), which is rapid and relatively inexpensive (27). MS-MCA is a reliable technique, and the results do not need to be verified by other techniques, such as is required for example for positive MSP results. The MS-MCA technique is based on the fact that the melting temperature of methylated and unmethylated alleles are different after modification of unmethylated cytosine and amplification, which converts methylated C:G base pairs to A:T base pairs with a lower melting temperature. The standard protocol for determination of methylation status MS-MCA stipulates that the oligonucleotide primers used to amplify the target nucleic acid are devoid of CpG dinucleotides to ensure that the primers does not discriminate between methylated an unmethylated target alleles. This constraint limits the primer binding sites to regions of the template without CpG dinucleotides, which can be very difficult or impossible to find within a CpG cluster in the region of interest. Furthermore, the unmethylated and methylated alleles are not equally efficient amplified. There is a significant bias towards the unmethylated allele in the PCR amplification (23).
Currently, no methylation detection method has been established for reliable, fast and cost-effective locus specific methylation testing that is readily applicable for both research and diagnostic settings. The research-based methods have various limitations and pitfalls and contradictory results can be obtained using different protocols, therefore none of them have found ready applicability in diagnostics (9).
A new more reliable method for promoter methylation analyses in clinical samples is needed.
The present invention offers a method for the determination of methylation status of a CpG-containing nucleic acid by nucleic acid amplification employing a novel design of primers, methylation-independent oligonucleotide primers, that allows for the use of only one set of primers to detect both alleles of a CpG-containing nucleic acid after it has been subjected to C to T conversion by conventional techniques.